The investigation of the effect of neural stem cells and hyaluronic acid hydrogel loded with tenascin C glycoprotein for reconstruction of sensorymotor cortex in a mouse model of photochemical ischemia

Abstract

In this study, the effect of three-dimensional scaffolding of hyaluronic acid modified with tenascin C glycoprotein was investigated in ischemic stroke. materials and methods: After the isolation and culture of neuronal stem cells, their phenotype was determined by flow cytometry analysis. Cell survival was assessed at different concentrations of Tenascin- C as well as in hyaluronic acid modified with Tenascin- C by MTT assay and respectively, DAPI and Acridine orange / Ethidium bromide staining were used to evaluate cell Homing and the number of living and dead cells in hyaluronic acid modified with Tenascin- C. Then, 7 days after induction of the ischemic model, cells labeled with DiI dye, hyaluronic acid biomaterial and Tenascin- C glycoprotein were injected into the stroke lesion in groups of mice (n = 10) by the stereotaxic method. 3 days after injection, DiI staining and Nestin expression were evaluated for neuronal stem cell survival and proliferation, and 28 days after injection, DiI staining and NeuN marker expression were evaluated for neuronal stem cell survival and differentiation by immunofluorescence staining. Also, on day 28, the mNSS behavioral test was used to evaluate sensory and motor functional improvement and crystal violet staining were used to evaluate the reduction of lesion volume. Results: In vitro study, MTT results showed the highest cell viability after 24 hours was at 100 nM Tenascin- C concentration. Also, MTT results after 24 and electron microscopy images after 72 hours of cell culture, respectively, showed that cell viability and adhesion were higher in the HA + TN-C group than in the HA group. The results of DAPI and AO / EB staining at 24, 48 and 72 hours after cell culture showed that although cell viability and the number of living cells increased in both groups, the percentage of living cells in the HA + TN-C group was higher than HA group. In vivo study, immunofluorescence staining on the third and 28th days after injection showed that the increase in proliferation and differentiation of neuronal stem cells was greater in NSCs + HA + TN-C group than other groups. Also, on the 28th day, in Crystal violet staining, the reduction in lesion volume in the NSCs + HA + TN-C group was higher than in the other groups and the behavioral test showed a significant difference performance improvement in this group compared to the control group.